Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica

The industrial yeast Yarrowia lipolytica secretes high amounts of an alkaline extracellular protease encoded by the XPR2 gene. The industrial use of the XPR2 promoter was however hindered by its complex regulation. We designed hybrid promoters, based on tandem copies of the XPR2 promoter UAS1 region. In contrast to native XPR2 promoter, these hybrid promoters were not repressed by the preferred carbon and nitrogen sources, nor by acidic conditions, and they did not require the presence of peptones in the culture medium. They exhibited a strong quasi-constitutive activity, similar when carried on either integrative or replicative plasmids. We used these hybrid promoters to direct the production of bovine prochymosin, using XPR2 secretion signals. The production of active chymosin was several fold higher than with previously available Y. lipolytica promoters (up to 160 mg/l). Integrative vectors based on the hybrid promoters, allowing the easy insertion of a heterologous gene and its expression or expression/secretion in Y. lipolytica, were designed. We also designed new Y. lipolytica recipient strains with good secreting abilities, able to grow on sucrose, and devoid of extracellular proteases. These new tools will add to the interest of Y. lipolytica as a host for heterologous protein production.     

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability

The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6 U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40 °C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30 min of incubation at 55 °C.     

Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica.

The XPR2 gene encoding an alkaline extracellular protease (AEP) from Yarrowia lipolytica was cloned, and its complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AEP consists of 297 amino acids with a relative molecular weight of 30,559. The gene codes for a putative 22-amino-acid prepeptide (signal sequence) followed by an additional 135-amino-acid propeptide containing a possible N-linked glycosylation site and two Lys-Arg peptidase-processing sites. The final Lys-Arg site occurs at the junction with the mature, extracellular form. The mature protease contains two potential glycosylation sites. AEP is a member of the subtilisin family of serine proteases, with 42.6% homology to the fungal proteinase K. The functional promoter is more than 700 base pairs long, allowing for the observed complex regulation of this gene. The 5' and 3' flanking regions of the XPR2 gene have structural features in common with other yeast genes.     

Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica

We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica. It was found to encode a 334-amino-acid precursor protein. The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC 3.1.1.3). The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by XPR6. Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme. Thus, the pro region does not inhibit lipase secretion and activity. However, it does play an essential role in the production of a stable enzyme. Processing was found to be correct in LIP2(A) (multiple LIP2 copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that XPR6 maturation was not limiting. No extracellular lipase activity was detected with the lip2 knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the LIP2 gene. Nevertheless, the lip2 KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in Y. lipolytica.     

Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.     

Role of the proregion in the production and secretion of the Yarrowia lipolytica alkaline extracellular protease

The yeast Yarrowia lipolytica secretes an alkaline extracellular protease (AEP). It is first synthesized as a precursor comprising a putative signal peptide, a stretch of 10 X-Ala or X-Pro sequences that are substrates for a dipeptidyl aminopeptidase, a large pro-region that contains a glycosylation site and two Lys-Arg sites that can be cleaved by a KEX2-like endoprotease and finally the mature protease itself. A defect in the XPR6 (KEX2-like) gene results in the secretion of an inactive proenzyme (Matoba, S., and Ogrydziak, D. M. (1989) J. Biol. Chem. 264, 6037-6043), showing that the proregion inhibits protease activity. To determine whether the proregion plays an additional role in protease secretion, we have generated deletions and point mutations in the corresponding region of the structural gene. In this paper we examine the effects of these mutations on AEP secretion and maturation and show that the proregion is essential for its secretion. All deletions affecting the proregion resulted in the intracellular accumulation of unprocessed precursors. Deletion of the glycosylation site in the proregion resulted in the production of an unglycosylated precursor that was secreted and matured correctly at 18 degrees C but accumulated in the cells at 28 degrees C. From these results, we propose that the AEP prosequence plays an additional essential role in guiding the proper folding of the protein into a conformation compatible with secretion.     

Extracellular RNase produced by Yarrowia lipolytica

Production of extracellular RNase(s) by Yarrowia lipolytica CX161-1B was examined in media between pHs 5 and 7. RNase production occurred during the exponential growth phase. High-molecular-weight nitrogen compounds supported the highest levels of RNase production. Several RNases were detected in the supernatant medium. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the RNases had estimated molecular weights of 45,000, 43,000, and 34,000. It was found that Y. lipolytica secretes only one RNase (the 45,000-molecular-weight RNase) and that the 43,000 and 34,000-molecular-weight RNases are degradation products of this RNase. The alkaline extracellular protease secreted by Y. lipolytica was shown to have a major role in the 45,000- to 43,000-molecular-weight conversion, and it was demonstrated that the 45,000-molecular-weight RNase could be purified from a mutant which does not produce the alkaline extracellular protease. Purification of the RNase from a wild-type strain resulted in purification of the 43,000-molecular-weight RNase. This RNase was a glycoprotein with a molecular weight of 44,000 as estimated by gel filtration, an isoelectric point of pH 4.8, and a pH optimum between 6.5 and 7.0.     

Use of ars18 based vectors to increase protein production in Yarrowia lipolytica.

The isolation of ars sequence from the yeast Yarrowia lipolytica has recently been reported (Fournier et al., 1991). Vectors containing ars18 have been used to increase homologous and heterologous protein production. Examples presented are the Yarrowia lipolytica alkaline extracellular protease (AEP), the porcine alpha 1-interferon and the bovine prochymosin. A 2- to 6-fold increase in the corresponding protein production was observed and in several cases it was established that it corresponded to the copy number of plasmid in the cell.     

Secretion of human blood coagulation factor XIIIa by the yeast Yarrowia lipolytica.

The industrial yeast, Yarrowia lipolytica, secretes high yields of an alkaline extracellular protease (AEP), which is synthesized as a preproprotein encoded by the XPR2 gene. We investigated the possibility of using this system for the secretion of human coagulation factor XIII subunit a (FXIIIa). This protein is naturally secreted in the plasma by an unknown, signal peptide-independent mechanism and has so far been found to be nonsecretable in yeast. We have designed six hybrid genes encoding fusion proteins between increasing portions of the AEP preprodomain and the precursor or mature forms of FXIIIa. All constructs directed translocation of the FXIIIa precursor into the endoplasmic reticulum. Transport of the translocated and core-glycosylated hybrid precursor to the Golgi apparatus appeared to be strongly rate limiting, and most of the precursors appeared to be partially proteolysed. One of these constructs directed the extracellular secretion of a low amount of hyperglycosylated FXIIIa. These results indicate that fusion to the yeast AEP signal peptide and dipeptide stretch allows FXIIIa to be translocated, albeit inefficiently, through the endoplasmic reticulum and to follow a classical secretory transit.     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

R.E. LEVIN AND R. WITKOWSKI. 1991. Yarrowia (Canadida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica , designated CL1 and CL2, with casin as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50.C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

Yarrowia (Candida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica, designated CL1 and CL2, with casein as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50 degrees C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0.     

强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能

地衣芽孢杆菌2709由于易于培养、GRAS状态和完善的蛋白质分泌能力,是已经投入工业生产碱性蛋白酶的菌株.为改善该菌株的发酵生产性能,提高菌体对培养基成分的利用和碱性蛋白酶产量,对菌株的胞外分泌酶系进行完善.利用同源重组机制,在基因组复制起始位点附近引入了来源于短小芽孢杆菌的木聚糖酶基因xynA和在复制起始位点中心对称...     

解脂耶氏酵母新型表达载体构建及癌基因rho在其中的表达

为了简化解脂耶氏酵母表达载体构建过程、消除抗生素污染,将mel基因(编码酪氨酸酶)作为新型报告基因用于构建新型酵母表达载体,利用组装PCR人工合成基因mel,并用重叠PCR将其与同源组成型强启动子p TEF、分泌性信号肽XPR2pre及强终止区LIP2t融合,构建新型胞外及胞内表达载体,并利用其在解脂耶氏酵母野生菌株中表达人源癌基因rho.成功获得mel全基因并将其与启动子、信号肽和终止区融合,得到融合片段TXML,用其替换原有表达载体的筛选标记基因ura3d4,构建得到新型胞外及胞内表达载体pINA1297-M和pINA1297-a-M,转化后的酵母阳性转化子性状明显,随后利用此新型表达系统获得可溶性异源蛋白Rho.首次实现了将mel作为一种便捷、价廉、无污染的新型筛选标记基因运用于非常规酵母表达系统中,更为mel在其它真核表达系统中的运用奠定了技术基础;获得的可溶性Rho蛋白可为研究其性质、结构、功能及与Rho癌基因家族其它成员的相互作用提供条件.     

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

Expression of POX2 gene and disruption of POX3 genes in the industrial Yarrowia lipolytica on the γ-decalactone production

The yeast Yarrowia lipolytica growing on methyl ricinoleate can produce γ-decalactone, the worthy aroma compound, which can exhibit fruity and creamy sensorial notes, and recognized internationally as a safe food additive. Unfortunately, the yield is poor because of lactone degradation by enzyme Aox3 (POX3 gene encoded), which was responsible for continuation of oxidation after C(10) level and lactone reconsumption. In this paper, we chose the industrial Y. lipolytica (CGMCC accession number 2.1405), which is the diploid strain as the starting strain and constructed the recombinant strain Tp-12 by targeting the POX3 locus of the wild type, one copy of POX3 was deleted by CRF1+POX2 insertion. The other recombinant strain Tpp-11, which was a null mutant possessing multiple copies of POX2 and disrupted POX3 genes on two chromosomes, was constructed by inserting XPR2+hpt into the other copy of POX3 of Tp-12. The growth ability of the recombinants was changed after genetic modification in the fermentation medium. The production of γ-decalactone was increased, resulting from blocking β-oxidation at the C(10) Aox level and POX2 overexpression. The recombinant strain Tpp-11 was stable. Because there was no reconsumption of γ-decalactone, the mutant strain could be grown in continuous fermentation of methyl ricinoleate to produce γ-decalactone.     

New types of RNA polymerase mutations causing temperature-sensitive sporulation in bacillus subtilis.

A single site mutant of Bacillus subtilis with a streptovaricin-resistant RNA polymerase has been isolated; this mutation caused temperature-sensitive sporulation, but had no effect on vegetative growth. The mutant (ts710) temperature-sensitive period irreversibly affected the middle and late stages of sporulation. Mutant cells grown at the nonpermissive temperature exhibited abnormal serine protease accumulation, serine esterase accumulation, alkaline phosphatase accumulation, RNA polymerase template specificity changes, and pulse-labeled RNA synthesis profiles. The accumulation of metal protease was not affected at the nonpermissive temperature. Attempts to isolate single site mutants which were streptolydigin-resistant, and temperature-sensitive for sporulation, were unsuccessful.     

Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase

Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.     

Regulation of extracellular alkaline protease activity by histidine in a collagenolytic Vibrio alginolyticus strain

Vibrio alginolyticus synthesized an inducible extracellular collagenase in a peptone medium during the stationary growth phase. These cultures also possessed extracellular alkaline serine protease activity. The alkaline protease activity did not require a specific inducer and it was produced in tryptone or minimal media. The collagenase was not produced in either the tryptone or minimal media. The alkaline protease activity was sensitive to catabolite repression by a number of carbon sources, including glucose, and by amino acids and ammonium ions. Cyclic AMP, dibutyryl cyclic AMP and cyclic GMP did not relieve catabolite repression. Histidine and urocanic acid stimulated the production of alkaline protease activity in tryptone and minimal media. Other compounds associated with the histidine utilization (hut) pathway did not increase alkaline protease activity. Histidine reversed the repression of alkaline protease activity by glucose of (NH4)2SO4 in minimal medium. Histidine and the compounds associated with the hut pathway inhibited collagenase production.